Contribution of a nuclear factor-kappaB binding site to human angiotensinogen promoter activity in renal proximal tubular cells

Abstract

Intrarenal angiotensinogen (AGT) is expressed highly in renal proximal tubular cells (RPTCs) and contributes to the regulation of intrarenal angiotensin II levels. Inhibition of nuclear factor (NF)-κB suppressed human (h)AGT expression in human RPTCs. However, the presence and localization of an NF-κB binding site in the hAGT promoter region have not been determined. Therefore, this study was performed to demonstrate that an NF-κB binding site in the hAGT promoter region contributes to hAGT promoter activity in human RPTCs. The hAGT promoter region was cloned from -4358 to +122 and deletion analysis was performed. A possible NF-κB binding site was removed from the hAGT promoter region (M1) and mutated (M2). Human RPTCs were transfected, and hAGT promoter activity was determined by luciferase assay. The identity of DNA binding proteins from binding assays were determined by Western blot. Progressive 5'-end deletions demonstrated removal of a distal promoter element in hAGT_-2414/+122 reduced promoter activity (0.61 ± 0.12, ratio to hAGT_-4358/+122). Inhibition of NF-κB suppressed promoter activity in hAGT_-4358/+122 (0.51 ± 0.14, ratio to control) and hAGT_-3681/+122 (0.48 ± 0.06, ratio to control) but not in the construct without the NF-κB binding site. Promoter activity was reduced in the domain mutants M1 (0.57 ± 0.08, ratio to hAGT_-4358/+122) and M2 (0.61 ± 0.16, ratio to hAGT_-4358/+122). DNA binding levels of NF-κB protein were reduced in M1. These data demonstrate the functional importance of an NF-κB binding site in the hAGT promoter region, which contributes to hAGT promoter activity in human RPTCs.

Figure 1

The hAGT promoter region constructs used in this study. A, Deletion mutant constructs of the hAGT promoter region. Thick horizontal lines represent hAGT promoter region sequences; open boxes, the pGL4.14 luciferase reporter vector sequence. B, Domain mutations of the possible NF-κB binding site in the hAGT promoter region. M1 indicates the mutant-1 with a 10-bp removal of the possible NF-κB binding site; M2, the mutant-2 with point mutations in the possible NF-κB binding site. Nucleotides: G, guanine; A, adenine; C, cytosine; T, thymine; R, purine (A or G); Y, pyrimidine (C or T); N, any nucleotide.

Figure 2

A, 5′-End deletion reduces hAGT promoter activity. Deletion of a distal region from −3681 to −2414 reduced hAGT promoter activity (n=6). B, Parthenolide treatment reduced hAGT promoter activity in promoter constructs possessing region −3681 to −2414 (n=6). Open bars represent control values; filled bars, 10 μmol/L parthenolide treatment for 24 hours. These data are expressed as luminometric values and represent means±SD. *P<0.05 vs hAGT_−4358/+122; †P<0.05 vs hAGT_−2414/+122; **P<0.05 vs controls without parthenolide treatment.

Figure 3

The possible NF-κB binding site contributes to hAGT promoter activity. A, Removal of the possible NF-κB binding site in M1 reduced hAGT promoter activity (n=6). B, Site-directed mutagenesis of the possible NF-κB binding site in M2 reduced hAGT promoter activity (n=6). These data are expressed as relative luminometric units compared with hAGT_−4358/+122 and represent means±SD. *P<0.05 vs hAGT_−4358/+122. WT indicates hAGT_−4358/+122; M1, hAGT promoter region construct lacking the possible NF-κB binding site; M2, hAGT promoter region construct with mutated possible NF-κB binding site.

Figure 4

Deletion of the possible NF-κB binding site reduces NF-κB binding activity to hAGT promoter. Removal of the NF-κB binding site in M1 reduced the binding of NF-κB protein to the hAGT promoter region compared with hAGT_−4358/+122. DNA (−) indicates no-DNA control; NC, negative control DNA lacking an NF-κB binding site; hAGT, hAGT_−4358/+122; M1, mutant-1.